Abstract/Project Summary Study of intracellular bacterial pathogens and the interactions with their host have revealed many interesting aspects of host-pathogen arms race. The gram-negative bacterium Legionella pneumophila (L.p.) causes a severe form of pneumonia known as Legionnaires disease and is particularly interesting as it manipulates several host traffic processes to establish its replicative niche in an endoplasmic reticulum (ER)- like vacuole. L.p. subverts host membrane transport pathways by injecting effector proteins via its type IV secretion system, Dot/Icm. Understanding how bacteria sabotage host vesicular transport processes and manipulate them to their own advantage provides invaluable insights into disease and mammalian cell biology. The unfolded protein response (UPR) is an important cytoprotective pathway in the ER that is manipulated by various pathogens. Interestingly, while previous work demonstrated that most pathogens induce the UPR, we have discovered that L.p. both activates and inhibits it. The UPR is sensed by three ER membrane sensors: ATF6, PERK and IRE1. IRE1 has a lumenal domain and cytoplasmic endoribonuclease and kinase domains. We have shown that two L.p. effectors belonging to the glucosyltransferase family, Lgt1 and Lgt2 (Lgt1/2), block the IRE1-mediated XBP1u mRNA splicing. Glucosyltransferases are common among pathogen toxins but an effect on mRNA splicing has never been observed. Along with the UPR?s canonical role of sensing unfolded protein stress in the ER, it has also been implicated in the innate immune response. Therefore, this novel IRE1 role has energized efforts to understand previously uncharacterized relationships between pathogens and the UPR. In the first aim, this proposal will determine the mechanism of IRE1 inhibition by L.p. glucosyltransferase effectors. In addition to IRE1, we have discovered that L.p. also activates ATF6. This leads to the upregulation of prototypical UPR transcripts. However, yet unknown L.p. effectors are able to block the translation of some of these genes. Our second aim proposes to identify the effectors and elucidate the mechanism of post-transcriptional repression of UPR target genes by L.p. Lastly, in our third and final aim, we propose to understand how L.p. activates the ATF6 pathway. Molecular dissection of this interaction will unravel novel mechanisms of bacterial pathogenesis and provide tools for probing and manipulating the UPR, which is implicated in numerous human diseases. We have assembled an exceptionally strong team of experts and compelling preliminary data that highlight our ability to accomplish our goals. We will combine the microbiology and molecular genetics expertise of the Mukherjee lab in L.p. biology with the cell biology expertise of the Walter lab to discover novel regulators of the UPR pathway.